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J. Agric. Food Chem. 2005, 53, 8586−8592
Influence of the Freshness Grade of Raw Fish on the Formation of Volatile and Biogenic Amines during the Manufacture and Storage of Vinegar-Marinated Anchovies SOFIÄA PONS-SAÄ NCHEZ-CASCADO, M. CARMEN VIDAL-CAROU,* ABEL MARINEÄ -FONT, AND M. TERESA VECIANA-NOGUEÄ S Departament de Nutricio´ i Bromatologia, Facultat de Farma`cia, Universitat de Barcelona, Avinguda Joan XXIII s/n, E-08028 Barcelona, Spain
Volatile and biogenic amines from three batches of anchovies, marinated in vinegar, were studied. The anchovies had been vacuum-packed and kept in refrigerated storage for 3 months. Trimethylamine and total volatile basic nitrogen levels were very low and constant throughout marinating and storage process: less than 1 and 10 mg/100 g, respectively. Certain amine levels, mainly those of tyramine and serotonin, increased slightly, particularly during storage. However, even the highest recorded levels were much lower than those considered to be hazardous for human consumption. To study the influence of raw material freshness in the amine profile, two laboratory trials using fresh and spoiled anchovies and simulating the industry standard marinating process were carried out. Levels for both volatile and biogenic amines were dependent on raw material quality, proving consistently higher in those deriving from nonfresh fish. Vinegar marinating leads to a decrease in the accumulation of amines in anchovy while their concentration in the vinegar solution increases due to the vinegar effect as solvent extractor. KEYWORDS: Anchovy; semipreserves; trimethylamine; total basic volatile nitrogen; biogenic amines; histamine
INTRODUCTION
Anchovy (Engraulis encrasicholus) is a pelagic fish belonging to the Engraulidae family. Anchovies caught off the Bay of Biscay and Mediterranean coasts are commonly consumed in Spain as fresh fish between May and October. However, up to 70% of capture is destined to the manufacture of semipreserves, mainly salted but also marinated in vinegar. Marinades are made by treating fresh or frozen fish, or portions of them, with edible acids (usually acetic acid) and salt, before the fish are placed in brine, sauces, creams, or oils (1). Marinating increases ionic strength and decreases pH, not only preventing microorganism growth but also involving changes in taste and in the textural and structural properties of fish (2). Although, in general, the quality of raw materials affects the quality of fish derivatives, few data on marinated fish are currently available. Trimethylamine (TMA-N) and total volatile basic nitrogen (TVB-N) have been proposed in several studies (3-5) as objective indices to evaluate the freshness of marine fish. TMA-N is a volatile basic compound that accumulates during fish spoilage from the reduction of trimethylamine oxide (TMA-O) by bacterial enzymes (6). Although TMA has been traditionally recognized as the compound mainly responsible for the characteristic fishy odor (7), it seems that other * Author to whom correspondence should be addressed (telephone +34 93 4024513; fax +34 93 403 59 31; e-mail
[email protected]).
compounds may be even more important (8). TMA-N, ammonia, and other basic compounds constitute TVB-N. Biogenic amines, which are basic organic compounds of low molecular weight, have also been proposed as indices of freshness in fish (9-11). Biogenic amines stem from decarboxylation of precursor amino acids by bacterial enzymes (12, 13). In fish marinades, acetic acid reduces bacterial growth and enzyme action, resulting in products with extended but limited shelf life. Some biogenic amine accumulation in marinades could be expected, as they are semipreserves wherein certain microorganisms or enzymes may remain active. In addition, acidic conditions make the tissue cathepsins more active, yielding higher amino acid release from proteins (14). Besides their use as freshness indices, biogenic amine levels can also warn consumers to potential health hazards related to fish consumption. Migraine headaches and histamine intoxication (HI) are directly produced by the intake of biogenic amines (15-17). Moreover, there are also indirect toxic effects related to biogenic amines, mainly tyramine, intake such as their interaction with monoamine oxidase inhibitor (MAOI) drugs (18). MAOI drugs block the oxidative deamination pathway of biogenic amines, giving rise to their accumulation and causing hypertensive crises, which can be mortal (19). Histamine, the most widely studied biogenic amine in fish, is the only amine for which legal limits have been established. The U.S. Food and Drug Administration (FDA) recommends
10.1021/jf050867m CCC: $30.25 © 2005 American Chemical Society Published on Web 09/30/2005
Amine Formation in Vinegar-Marinated Anchovies determining other biogenic amines together with histamine (20) because they are known to aggravate histamine toxicity (21). Fish marinades incorporating vinegar have been popular in Mediterranean countries since antiquity, but their consumption has historically been limited to the fishing season. Today, however, anchovies marinated in vinegar are also manufactured in factories, making them available to consumers year round. Although several studies address biogenic amine content and evolution in salt-ripened anchovies or marinades from other fish species (14, 22-25), none examine anchovies marinated in vinegar. The aim of the present work was to study the usefulness of volatile and biogenic amines as quality indices of raw materials in anchovies marinated in vinegar. Samples of raw materials and their corresponding end products were analyzed. As end products are not thermally treated, possible changes stemming from the storage process were also examined. In addition, a laboratory trial simulating the industrial marinating process was carried out to investigate the effects of raw material quality on the amine profile. MATERIALS AND METHODS Factory Samples. Samples were provided by an artisan producer who elaborates anchovies marinated in vinegar as follows: Immediately upon arrival, the fresh fish (E. encrasicholus) is cleaned, beheaded, gutted manually, and then immersed in a vinegar solution (20% acetic acid v/v). A maceration period of 2 weeks is maintained, during which fish remain under refrigeration. Anchovies are then manually filleted, immersed in oil, and vacuum-packed. Three different batches were studied, including samples of fish and vinegar of both raw materials and end products. To determine whether changes in biogenic amines occurred during storage, end product samples were divided into two groups: samples of the first group were analyzed upon arrival at the laboratory, and samples of the second group were analyzed after a refrigerated storage period of 3 months, which is the manufacturer’s recommended shelf life. Laboratory Trial Samples. Anchovies (2-3 kg) caught off the Mediterranean coast were obtained from a fish market in Barcelona and marinated at the laboratory following the same procedure used by the artisan producer. Individual fish weighed 11-16 g and were 1015 cm in length. Upon arrival at the laboratory, the freshness state of anchovies was assessed using the European Union (EU) grading scheme for fatty fish (26) and resulted in a grade of Extra (E). Whole fish were divided in two batches to proceed with marination. (a) Batch F (fresh) anchovies were immediately gutted, beheaded, immersed in a 40:60 mixture of wine vinegar/water, and kept refrigerated for 2 weeks. (b) Batch S (spoiled) anchovies were kept at room temperature until they met C grade of the EU criteria (26), which means spoiled fish, unacceptable for human consumption. Then fish were gutted, beheaded, and marinated in an identical way to those of batch F. Fish and vinegar samples were taken at time 0 (nonmarinated fish and newly acquired vinegar) and after 3, 8, and 14 days of marinating. At each sampling time, 10-12 anchovies, selected at random, were collected and their flesh was homogenized for determination of TMAN, TVB-N, and biogenic amine levels. This laboratory trial was performed in duplicate. Chemical Analysis. TMA-N and TVB-N levels were measured by flow injection-gas diffusion following extraction from fish with 7.5% trichloroacetic acid (27). Afer alkalinization with NaOH, volatilized amines induced a change in the color of the blue bromothymol solution proportional to the amount of TMA-N or TVB-N, which was detected at λ 635 nm. Quantification was carried out on the basis of a calibration curve calculated from the response given by standard solutions of TMA (for TMA-N) and ammonium chloride (for TVB-N). Standards were obtained from Panreac Quimica SA (Barcelona, Spain).
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Biogenic amine levels were analyzed by high-performance liquid chromatography (HPLC) from a 0.6 N perchloric acid extract according to the method of Veciana-Nogue´s et al. (28). Briefly, the method involves the separation of ion pairs formed between biogenic amines and octanosulfonic acid present in the mobile phase on a reverse phase column (Nova Pack C18, Waters Cromatografı´a, S.A.). A postcolumn derivatization employing o-phthalaldehyde (OPA) is followed by an spectrofluorometric detection (28). The elution gradient allowed the same-run separation of 12 biogenic amines, the identification of which was done according to the retention times obtained for the standards (Figure 1). Quantification of each amine was performed from the corresponding calibration curve calculated after the injection of solutions at different concentration (from 0.25 to 10 mg/L) of amine standards (Sigma, St. Louis, MO), with a satisfactory precision (relative standard deviation below 5%) and sensitivity (limit of quantification below 0.5 mg/kg for all amines). All chemical analyses were carried out in duplicate. Statistical Analysis. Statistical analysis of data was conducted using SPSS 11.0 for Windows software (SPSS Inc., Chicago, IL). RESULTS AND DISCUSSION
Table 1 shows the TMA-N, TVB-N, and biogenic amine levels for factory samples both before and following marinating, as well as after 3 months of storage. As expected, TMA-N and TVB-N were not found in fresh vinegar. In fresh and just-packed marinated anchovies, TMA-N content was
